Methods and compositions for tagging and analyzing samples

This application is a continuation of U.S. patent application Ser. No. 16/884,518 filed May 27, 2020, and issued as U.S. Pat. No. 11,161,087 on Nov. 2, 2021, which is a continuation of U.S. patent application Ser. No. 16/043,060 filed Jul. 23, 2018, and issued as U.S. Pat. No. 10,722,858 on Jul. 28, 2020, which is a continuation of U.S. patent application Ser. No. 14/776,177 filed Sep. 14, 2015, and issued as U.S. Pat. No. 10,058,039 on Aug. 28, 2018, which is a national stage application under 35 U.S.C. § 371 of International Patent Application No. PCT/US2014/029393 filed Mar. 14, 2014, which claims priority to U.S. Provisional Application No. 61/806,143, filed Mar. 28, 2013, and U.S. Provisional Application No. 61/801,785, filed Mar. 15, 2013, both of which applications are incorporated herein by reference in their entireties.

The instant application contains a Sequence Listing which has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on 16 Oct. 2025, is named 27050024US04SEQL.txt and is 5,939 bytes in size.

The analysis of nucleic acids and proteins in biological samples is an essential element of molecular biology. The ability to detect, discriminate, and utilize genetic and proteomic information allows sensitive and specific diagnostics as well as treatment.

The present invention provides for rapid tagging and analysis of nucleic acids and protein at the single cell level.

The present invention provides methods of tagging target oligonucleotides that include steps of (a) partitioning DNA into a plurality of compartments; (b) performing an in vitro transcription reaction on the DNA within the compartments, thereby obtaining compartments comprising RNA; (c) merging the interior of the compartments comprising RNA with the interior of a set of compartments comprising target oligonucleotides; (d) hybridizing the RNA to the target oligonucleotides; and (e) performing a reaction to attach a sequence corresponding to the RNA to the target oligonucleotides.

In some embodiments, the DNA is double stranded.

In some embodiments, the compartments are droplets within an oil-and-water emulsion.

In some embodiments, the target oligonucleotides include at least one target oligonucleotide comprising a cell tag and a molecule tag.

In some embodiments, the target oligonucleotides are DNA.

In some embodiments, the method may further include a step of partitioning the target oligonucleotides into the set of compartments before the merging of step (c).

In some embodiments, the method may further include a step of partitioning a set of cells into the set of compartments and lysing the cells in order to liberate cellular oligonucleotides before the merging of step (c). For example, the cellular oligonucleotides are the target oligonucleotides. Alternatively, the cellular oligonucleotides are cellular mRNA.

In some embodiments, the method may further include a step of conducting a reverse transcription on the cellular mRNA to generate cellular cDNA. For example, the reverse transcription reaction is performed with a primer specific for a region of the genome. The region of the genome can be an immunoglobulin gene or a T-cell receptor gene.

For example, the reverse transcription reaction can be conducted within the set of compartments prior to the merging step.

Alternatively, the reverse transcription reaction can be conducted within the merged compartments.

In some embodiments, the target oligonucleotides are cellular cDNA.

In some embodiments, the reaction is a Rapid Amplification of cDNA Ends (RACE) reaction.

In some embodiments, the DNA is conjugated to a solid support. For example, the solid support is a bead.

The present invention also provides methods that include steps of (a) providing a plurality of beads comprising a plurality DNA oligonucleotides; (b) providing a plurality DNA oligonucleotides comprising a primer sequence, a universal sequence an adapter sequence and a cellular tag; (c) merging the beads of step (a) and the oligonucleotides of step (b) into a plurality of compartments such that each compartment comprises a single bead and a single oligonucleotide; (d) performing an amplification reaction on the oligonucleotides within the compartments, thereby obtaining a plurality of DNA oligonucleotides comprising the primer sequence, the universal sequence, the adapter sequence and the cellular tag; (e) performing an in vitro transcription reaction on the DNA within the compartments, thereby obtaining compartments comprising RNA the primer sequence, the universal sequence and cellular tag; (f) merging the interior of the compartments comprising RNA with the interior of a set of compartments comprising target oligonucleotides; (g) hybridizing the RNA to the target oligonucleotides; and (h) performing a reaction to attach a sequence corresponding to the RNA to the target oligonucleotides.

In some embodiments, the plurality of oligonucleotides on the bead includes a molecule tag.

The present invention further provides methods of tagging target oligonucleotides that include steps of (a) isolating a plurality of mRNA from a biological sample comprising a plurality of cell types; and (b) performing reverse transcription of the mRNA using a primer specific for the target oligonucleotide and a template switching oligonucleotide comprising a molecule tag, a universal sequence, and an adapter sequence to produce tagged target cDNA.

In some embodiments, the target cDNA is tagged at the 3′end.

In some embodiments, the target oligonucleotide is an immunoglobulin or T-cell receptor.

In some embodiments, the adapter sequence is specific to a sequencing platform.

In some embodiments, the molecule tag is an oligomer. For example, the oligomer is a randomer.

In some embodiments, the randomer is at least a 9 mer.

In some embodiments, the method further includes a step of amplifying the target cDNA using the universal sequence and a primer specific for the target oligonucleotide.

In some embodiments, the method may further include a step of sequencing the amplified cDNA.

Also provided are methods of determining the immune repertoire in a subject by (a) isolating a plurality of mRNA from a biological sample comprising a plurality of cell types; (b) performing reverse transcription of the mRNA using a immunoglobulin or T-cell receptor specific primers and a template switching oligonucleotide comprising a molecule tag, a universal sequence and an adapter sequence to produce molecule tagged immunoglobulin or T-cell receptor cDNA; (c) amplifying the cDNA using the universal sequence and a primer specific for the target oligonucleotide; (d) sequencing the cDNA to produce a plurality of sequencing reads; (e) grouping the sequence reads with the same molecule tag and clustering the sequences within the same group; and (f) building a consensus sequence for each cluster to produce a collection of consensus sequences wherein the consensus sequence is used to determine the diversity of the immune repertoire. In some embodiments, the target cDNA is tagged at the 3′end.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

I. Overview

This disclosure provides methods and compositions for tagging molecules and subsequent analysis of the tagged molecules. In some cases, the disclosure provides methods and compositions for partitioning analytes (e.g., cells, polynucleotides) into individual partitions (e.g., droplets, wells, spots on arrays, etc.) and also provides methods and compositions for labeling the analytes within the partition.

In some cases, this disclosure generally relates to obtaining a sample (e.g. blood, saliva, tissue, cells) from a subject [] (e.g. human, animal, plant, fungus, bacteria, population of cells, biofilm), partitioning and labeling the sample components with tagging molecules [], processing and analyzing the labeled sample components [], and/or reporting results from the analysis []. Cellular components such as DNA and RNA, individual cells or a population of cells may be partitioned. Cell population can include cells of similar function, such as for example, immune cells (e.g. B-cells or T-cells), cancer cells, or nerve cells. Moreover, a population of cells may be partitioned into cell partitions [] and, separately, a population of beads with surface-bound oligonucleotides (e.g., DNA, dsDNA) may be partitioned within bead partitions []. The individual cells may be lysed within the cell partitions []. In some cases, the oligonucleotides are transcribed from the beads [] such that RNA transcripts are produced in the bead partitions. In some cases, the RNA transcripts are reverse-transcribed into cDNA within the bead partitions; in some cases, the RNA transcripts are reverse-transcribed into cDNA within the bead partitions at a later step, such as after the contents of the bead partitions (e.g., droplets) are merged with the contents of another partition (e.g., a different droplet). The cell partitions may be combined with the bead partitions on a partition-by-partition basis []. Tagging reactions may then be conducted within the combined partitions in order to tag the cellular mRNA (or cDNA derived therefrom) with the oligonucleotides derived from the beads []. The tagged products may then be pooled, amplified, and sequenced [].

The methods and compositions provided in this disclosure allow for tracking information sources and preserving the heterogeneity of information in a sample as it is analyzed. By labeling a sample at the individual component level, the resolution of the information can be maintained at the individual component level throughout the stages of analysis, regardless of subsequent merging or combining of the components. For example, a sample containing multiple cell types can be partitioned to a single cell type. Cell-types that can be partitioned into a single cell type include for example immune cells such as B-cells or T-cells. Alternatively, a sample containing multiple cell types can be partitioned into individual partitions containing single cells. By labeling the information-bearing molecules (e.g. DNA, RNA, protein) from a single cell type or a single cell, the individual partitions can then be merged for further analysis without loss of the single cell-level information resolution. The methods and compositions provided herein may also include additional labels, such as labels that enable quantification of the individual molecules within a partition. For example, a single partition may contain numerous unique labels, each with a different sequence that can be used to tag and quantify individual molecules within a partition.

The methods, compositions and kits provided herein are broadly applicable to a variety of life science-related fields, including biomedical research, drug discovery and development, and clinical diagnostics. Potential applications include gene expression profiling at the single cell type or single cell level for the detection and/or monitoring of cancer, autoimmune disease, viral infection, organ transplant rejection, and other diseases or disorders. The present disclosure may also be used to (a) analyze the immune repertoire of a subject, such as a subject with a particular disease or disorder; (b) elucidate intracellular signaling pathways; (c) validate therapeutic targets for drug discovery and development; and (d) identify or detect biomarkers, particularly biomarkers related to normal or diseased biological states. The present disclosure may also be used to analyze circulating cell-free DNA or RNA in order to predict, monitor, detect and/or diagnose conditions or diseases, including organ rejection.

The methods and compositions disclosed herein offer several important advantages over existing techniques for monitoring gene expression in cells or tissue. Importantly, the methods and compositions provided herein enable the monitoring and detection of gene expression in single cells, thereby eliminating the systematic errors and noise that may arise due to sampling of heterogeneous cell populations when collecting data using conventional techniques. The existence of heterogeneous cell populations in test samples may arise, for example, from asynchronous cell division in populations of cultured cells; or, in some cases, heterogeneity may be due to mixtures of different cell types present in tissue samples, biofilms, bioreactor samples, blood samples, biopsy samples or other complex samples. Another important advantage of the methods and compositions disclosed herein is the potential for eliminating or reducing errors caused by PCR amplification bias, for example, through the use of molecular tags that label different molecules within a sample. For example, if, when analyzed, the same molecular sequence is found to have two different tags, this may indicate that there were two copies of the molecule within the partition. This information may also be useful to discount results due to amplification errors. A third important advantage of the methods and compositions disclosed herein is the potential for expanding the range of biomarkers used to sort and classify cells. In addition to targeting gene sequences that code for the extracellular protein markers, the approach described herein enables the use of intracellular markers, for example gene sequences coding for transcription factors or cytokines, for cell sorting and classification in order to facilitate correlations between gene expression and cell function. The present disclosure thus offers an approach for obtaining higher quality genomic data from biological samples, and thus the potential for developing better therapeutics and improved detection of disease.

II. Assays

A. Labeling within Partitions

This disclosure provides methods and compositions for tagging analytes at a single component level, such as at the level of a single cell type or single cell. In some cases, analytes are partitioned into a set of partitions; labels are partitioned into a separate set of partitions, and the contents of individual partitions within each set are combined to enable labeling of the analyte.

Partitioning Analytes

A sample comprising analytes (e.g., cells) can be partitioned into a set of individual partitions (e.g., droplets or wells). In some cases, a partition within the set of individual partitions contains at most one analyte. In some cases, a partition within the set of individual partitions contains at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 analytes. In some cases, a partition within the set of individual partitions contains, on average, one analyte. In some cases, a partition within the set of individual partitions contains, on average, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 analytes, or more. In some cases, the set of individual partitions comprises empty partitions. Often, the set of individual partitions comprises some empty partitions and some partitions comprising analytes (e.g., at most one analyte, at most two analytes, etc.). In some cases, an analyte comprises a plurality of components (e.g., a plurality of molecules). In some cases, a technique is applied to ensure that all of the partitions comprise at most one analyte; for example, the empty partitions (e.g., droplets) may be sorted out by a flow sorter.

Partitioning Tags

Tags or solid supports (e.g., beads) conjugated to tags can be partitioned into a set of individual partitions. In some cases, a partition within the set of individual partitions contains at most one tag or solid support (e.g., a bead) conjugated to a tag. In some cases, a partition within the set of individual partitions contains at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 tags or solid supports (e.g., beads) conjugated to a tag. In some cases, a partition within the set of individual partitions contains, on average, one tag or solid support (e.g., bead) conjugated to a tag. In some cases, a partition within the set of individual partitions contains, on average, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 tags or solid supports (e.g., beads) conjugated to a tag. In some cases, the set of individual partitions comprises empty partitions. Often, the set of individual partitions comprises some empty partitions and some partitions comprising a tag (e.g., at most one tag, at most two tags, etc.). In some cases, a tag comprises a plurality of components (e.g., a plurality of molecules). In some cases, a technique is applied to ensure that all of the partitions comprise at most one tag or bead; for example, the empty partitions (e.g., droplets) may be sorted out by a flow sorter.

Labels or tags may be added to the contents of individual partitions in order to enable later identification of the particular analyte (e.g., cell) that is the source of a particular component (e.g., molecule). In some cases, the labels or tags are partitioned into a set of partitions; the partitions may then be used to label the partitioned analytes (e.g., cells).

The labels or tags may be conjugated to beads, for example, which are partitioned into the set of partitions (e.g., droplets). In some cases, material for generating labels or tags is partitioned into a set of partitions; the tags are then produced in the individual partitions and later used to label the analytes. For example, DNA tags (e.g., free DNA tags, DNA tags conjugated to beads) may be partitioned into individual partitions and then subsequently subjected to a polymerase chain reaction (PCR) to produce copies of the tags in solution within the partitions. The tags in solutions may then be used to directly label an analyte, or as a template that is used to label the analyte in a subsequent reaction. In some cases, the DNA tags (are subjected to an in vitro transcription reaction in order to produce RNA tags within the partitions. The RNA tags in solutions may then be used to directly label an analyte, or as a template that is used to label the analyte in a subsequent reaction such as a RACE reaction.

Intra-Partition Tagging

In some cases, the contents of the analyte and tag partitions described herein can be combined in order to facilitate labeling on a per-analyte (e.g., per-cell) basis. For example, if the partitions are droplets, individual droplets from an analyte set of droplets can be merged with individual droplets from a tag set of droplets in order to facilitate labeling of the analytes. Methods of combining contents of partitions are described elsewhere herein.

In some cases, a sample containing cells can be partitioned into individual partitions, each partition comprising cell(s). Tags can be applied to the analyte components (e.g., DNA, RNA, etc.) within a partition, so that each component within a partition is labeled with the same tag, such as a cell tag capable of identifying a particular cell. In some cases, tags can be applied to the analyte components (e.g., DNA, RNA, etc.) within a partition, so that a portion of the components within a partition is labeled with the same tag, such as a cell tag capable of identifying a particular cell. The tags may comprise a molecule tag, where each tag within a partition comprises a different molecule tag. In some cases, an individual tag may comprise both a cell tag and a molecule tag. The components within a partition may be labeled with tags, so that each analyte or analyte component in a partition is labeled with a different molecule tag label. The components within a partition may be labeled with tags, so that each analyte or analyte component in a partition is labeled with an identical cell tag. The components within a partition may be labeled with tags, so that each analyte or analyte component in a partition is labeled with a different molecule tag and an identical cell tag.

depicts exemplary methods provided herein. In some cases, a solution comprising beads conjugated to nucleic acids [] is partitioned into droplets []. In some cases, the droplets contain at most one bead. The nucleic acids may be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA, or a combination DNA and RNA. The nucleic acids may comprise a unique tag sequence (e.g. a 9-mer randomer). By “randomer” as used herein it is meant an oligonucleotide with randomly synthesized bases. When the beads are conjugated to dsDNA, in vitro transcription [] may then be used to produce RNA transcripts from the tag dsDNA conjugated to the beads []. The oligonucleotides can be template switching oligonucleotides. In some cases, a solution comprising cells [] is partitioned into droplets []. In some cases, the droplets contain at most one cell. Cells may be lysed [] Lysis can be performed by adding a lysis solution, buffer, or detergent to the cell, by sonication, by shear, by freezing and thawing, by heating, by electrical lysis, by grinding, or by any other appropriate method. Addition of lysis solution, buffer, or detergent can be performed by droplet injection, by droplet merging, or by any other appropriate method.

Droplets containing cell lysate [] may then be merged [] with droplets containing amplified oligonucleotides. Alternatively, the material within the droplets may be lysed following merging. Droplet merging may be conducted by any appropriate means, including passive droplet merging (e.g. at a microfluidic junction) and active droplet merging (e.g. electric, magnetic, thermal, or optical means). Alternatively, cell lysis may be conducted after droplet merging. Reverse transcription [] with oligonucleotides and cell lysate may then be performed in the merged droplet [] to produce cDNA [] from a single cell tagged with the same unique tag sequence.

In some cases, oligonucleotides derived from many cells may be tagged in parallel, for example on a high-throughput basis. A solution comprising beads with surface-bound nucleic acids and a solution comprising cells may each be partitioned into droplets. In some cases, the bead droplets ([], [], []) comprise a bead ([], [], []) and the cell droplets ([], [], []) comprise a cell ([], [], []). The nucleic acids may be double-stranded DNA, single-stranded DNA, RNA, or a combination DNA and RNA. The nucleic acids may comprise a unique tag sequence (e.g., a 9-mer randomer). In vitro transcription may then be used to amplify oligonucleotides with a tag sequence ([], [], []). The oligonucleotides can be template switching oligonucleotides. Cells may be lysed. Lysis can be performed by adding a lysis solution, buffer, or detergent to the cell, by sonication, by shear, by freezing and thawing, by heating, by electrical lysis, by grinding, or by any other appropriate method. Addition of lysis solution, buffer, or detergent can be performed by droplet injection, by droplet merging, or by any other appropriate method. Droplets containing lysate ([], [], []) may then be merged with droplets containing amplified oligonucleotides. Droplet merging may be conducted by any appropriate means, including passive droplet merging (e.g., at a microfluidic junction) and active droplet merging (e.g. electric, magnetic, thermal, or optical means). Alternatively, cell lysis may be conducted after droplet merging. Reverse transcription with oligonucleotides and cell lysate may then be performed in the merged droplets ([], [], []) to produce cDNA ([], [], []), where the cDNA in each droplet is from a single cell and is tagged with the same unique tag sequence. After labeling, the cDNA from all cells may be pooled []. The number of cells may be at least 10, at least 100, at least 1000, at least 10,000, or at least 100,000. PCR may then be conducted on the pooled cDNA []. The PCR may specifically target genes or regions of interest for sequencing. The genes or regions of interest may comprise immunoglobulin heavy chain (IgH), immunoglobulin light chain (IgL), Tcell receptor beta (TCRb), T-cell receptor-alpha (TCRa), or immune cell markers. Amplified DNA may then be sequenced []. Information from sequencing may then be demultiplexed [] based on the tag sequences.